CA2165679A1 - Anti-sickling hemoglobin - Google Patents

Abstract

Disclosed are anti-stickling human hemoglobins for use as sickle cell anemia therapeutics.

Description

WO95/~657 2 1 6 5 6 7 9 PCT~S94/06901 ANTI-SICKLING HEMOGLOBIN
Background of the Invention This invention relates to recombinant anti-5 sickling hemoglobins suitable for use as therapeutics forthe treatment of sickle cell anemia.
The gene that e~CoAec hemoglobin S (the defect leading to sickle cell anemia) is inherited as an autosomal trait and occurs in the heterozygous conditi-on 10 as the sickle trait in 8-10% of black persons in the Unites States. Two major clinical features characterize sickle cell anemia: (1) chronic hemolysis that is stable and only moderately debilitating, and (2) acute, episodic vaso-occlusive crises that cause organ failure and 15 account for most of the mortality and morbidity associated with ~he ~ se.
The molecular basis for sickle cell disease is an A to T transversion in the 6th codon of the human ~-globin gene. This simple transversion changes a polar 20 glutamic acid residue to a non-polar valine (Ingram et al., Nature 178:792, 1956; Ingram et al., Nature 180:326, 1957) in the ~-globin polypeptide and, thus, drastically decreases the solubility of this hemoglobin (termed Hb S). When the intracellular concentration of Hb S is high 25 and the partial pressure of oxygen is low in the capillary beds, the non-polar valine, which is on the surface of the hemoglobin molecule, interacts wit~ two other non-polar residues on the surface of a secQn~
hemoglobin molecule, and initiates aggregation (Padlan et 30 al., J. Biol. Chem. 260:8280-8291, 1985; Wish~e~ et al., J. Mol. Biol. 98:179-194, 1975). Once approximately 10 hemc~:lobin monomers interact, long polymers rapidly accumulate, and complex 14-stranded fibers are formed (Crepeau et al., Nature 274:616-617, 1978; Dykes et al., 35 J. Mol. Biol. 130:451-472, 1979; Eaton et al., Blood WO 951~657 2 1 6 5 6 7 9 PCT~S94/06901 70:1245-1266, 1987; Hofrichter et al., Proc. Natl. Acad.
Sci USA 71:4864-4868, 1974). The formation of these fibers reduces the flexibility of red blood cells and leads to the occlusion of small capillaries.
5 Intracellular fiber formation also results in erythrocyte membrane damage and increased red cell lysis (Noguchi et al., Blood 58:1057, 1981; Brittenham et al., Blood 65:183, 1985). The ensuing ~; c~c~ is characterized by a chronic hemolytic anemia with ep;coAeC of severe pain, 10 and tissue damage that can result in stroke, kidney failure, heart disease, infection, and other complications (Bunn et al., Hemoqlobin: Molecular.
Genetic. and Clinical AsPects. (W.B. Saunders, Philadelphia, 1986)).

SummarY of the Invention In one aspect, the invention features recombinant human hemoglobin with anti-sickling activity.
Preferably, such anti-sickling hemoglobin is derived from ~-globin. In preferred embodiments, the anti-sickling 20 hemoglobin includes a mutation which disrupts the hydrophobic pocket formed by ~-globin amino acids phenyl~l~n;n~ 85 and leucine 88, but which leaves intact the correct positioning of the heme moiety. Preferred anti-sickling human hemoglobins include: (a) a glutamine 25 residue at ~-globin amino acid 87; (b) a lysine residue at ~-globin amino acid 87; (c) a lysine residue at ~-globin amino acid 80; (d) an ~lAnine residue at ~-globin amino acid 22; (e) a glutamine residue at ~-globin amino acid 87 and an ~l~nine residue at ~-globin amino acid 22;
(f) a lysine residue at ~-globin amino acid 87 and an alanine residue at ~-globin amino acid 22; (g) a lysine residue at ~-globin amino acid 80 and an alanine residue at ~-globin amino acid 22; (h) a lysine residue at ~-globin amino acid 108; (i) a lysine residue at ~-globin W095/00657 2 1 6 5 6 7 9 PCT~S94/06901 amino acid 108 and an alanine residue at ~-globin amino acid 22; (j) a lysine residue at ~-globin amino acid 108 and a glutamine residue at ~-globin amino acid 87; (k) a lysine residue at ~-globin amino acid 108 and a lysine 5 residue at ~-globin amino acid 87; (l) a lysine residue at ~-globin amino acid 108 and a lysine residue at ~-globin amino acid 80; (m) a lysine residue at ~-globin amino acid 108, an AlAn;ne residue at ~-globin amino acid 22, and a glutamine residue at ~-globin amino acid 87;
(n) a lysine residue at ~-globin amino acid 108, an AlAn;ne residue at ~-globin amino acid 22, and a lysine residue at ~-globin amino acid 87; (o) a lysine residue at ~-globin amino acid 108, an alanine residue at ~-globin amino acid 22, and a lysine residue at ~-globin 15 amino acid 80; (p) a glutamic a-id residue at ~-globin amino acid 95; (q) a glutamic acid residue at ~-globin amino acid 95 and an AlAnine residue at ~-globin amino acid 22; (r) a glutamic acid residue at ~-globin amino acid 95 and a glutamine residue at ~-globin amino acid 20 87; (s) a glutamic acid residue at ~-globin amino acid 95 and a lysine residue at ~-globin amino acid 87; (t) a glutamic acid residue at ~-globin amino acid 95 and a lysine residue at ~-globin amino acid 80; (u) a glutamic acid residue at ~-globin amino acid 95, an AlAnine 25 residue at ~-globin amino acid 22, and a glutamine residue at ~-globin amino acid 87; (v) a glutamic acid residue at ~-globin amino acid 95, an AlAnine residue at ~-globin amino acid 22, and a lysine residue at ~-globin amino acid 87; (w) a glutamic acid residue at ~-globin 30 amino acid 95, an AlA~ residue at ~-globin amino acid 22, and a lysine residue at ~-globin amino acid 80; (x) an aspartic acid residue at ~-globin amino acid 16; (y) an aspartic acid residue at ~-globin amino acid 16 and an alanine residue at ~-globin amino acid 22; (z) an 35 aspartic acid residue at ~-globin amino acid 16 and a WO95/~K~7 2 1 6 5 6 7 9 PCT~S94/06901 glutamine residue at ~-globin amino acid 87; (a') an aspartic acid residue at ~-globin amino acid 16 and a lysine residue at ~-globin amino acid 87; (b') an aspartic acid residue at ~-globin amino acid 16 and a 5 lysine residue at ~-globin amino acid 80; (c') an aspartic acid residue at ~-globin amino acid 16, an Al~n;ne residue at ~-globin amino acid 22, and a glutamine residue at ~-globin amino acid 87; (d') an aspartic acid residue at ~-globin amino acid 16, an 0 A 1 Ani ne residue at ~-globin amino acid 22, and a lysine residue at ~-globin amino acid 87; (e') an aspartic acid residue at ~-globin amino acid 16, an alanine residue at ~-globin amino acid 22, and a lysine residue at ~-globin amino acid 80. Alternatively, the anti-sickling human 15 hemoglobin may include an arginine residue at ~-globin amino acid 48.
The anti-sickling hemoglobin of the invention is preferably enco~ by purified DNA, for example, purified DNA which includes a hemoglobin sequence encoding any of 20 the above-listed anti-sickling hemoglobins of the invention.
~ inally, the invention features methods for correcting a sickle defect in a mammal by gene therapy.
This method involves administering to the mammal a 25 purified nucleic acid enco~i~g a recombinant anti-sickling hemoglobin of the invention, the anti-sickling hemoglobin nucleic acid being positioned for expression in the mammal. In preferred methods, the anti-sickling hemoglobin-enco~i~g nucleic acid is delivered to the 30 mammal as part of a viral vector and is delivered to the mammal'æ bone marrow. Preferred viral vectors include, but are not limited to retroviral and adeno-associated viral vectors, and any modified versions of these vectors.

WO95/006~7 2 1 6 5 6 7 9 PCT~S94/06901 The term "recombinant", as used herein, means expressed from an isolated or purified DNA molecule. The recombinant anti-sickling hemoglobins described herein are produced by directed modifications (e.g., by site 5 directed or PCR mutagenesis) of such an isolated DNA
molecule.
The term "purified DNA", as used herein, means DNA
that is not immediately contiguous with both of the coding se~l~nceC with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally occurring genome of the organism from which the DNA was derived. The term therefore includes, for example, a recombinant DNA molecule which is incorporated into a vector, e.g., an autonomously replicating plasmid 15 or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is a 20 part of a hybrid gene ~nco~ing additional polypeptide sequences.
The term "human hemoglobin", as used herein, means a molecule whose amino acid sequence at least in part corresponds to the amino acid sequence of a naturally-25 occurring human hemoglobin molecule, whether mutated orwild-type.
The term "anti-sickling", as used herein, means capable of interfering with the aggregation of hemoglobin into 14-stranded hemoglobin molecules characteristic of 30 Hb S hemoglobin and resulting in sickle cell anemia (as described herein). Preferably, the anti-sickling molecules of the invention have approximately the same anti-sickling properties as fetal Hb (i.e., ~2~2) hemoglobin (e.g., as measured by in vitro solubility 2 1 6 5 6 7 9 PCT~S94/06901 assays, e.g., the assay of Benesch et al., J. Biol. Chem.
254:8169, 1979).
The term "Hb S hemoglobin" as used herein means that hemoglobin which aggregates into 14-stranded fibers 5 at high intracellular concentrations and low partial pressure; such Hb S hemoglobin has an A to T transversion in the 6th codon of the human ~-globin gene.
The term "positioned for expression" means that the DNA molecule is positioned adjacent to DNA sequences 10 which direct transcription and translation of the sequence (i.e., f acilitates the production of, e.g., anti-sickling hemoglobin).
Unless defined otherwise, all technical and scientific terms used herein have the same me~ning as 15 commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials 20 are now described. All publications mentioned herein are incorporated by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are st~nA~rd methodologies well known to one of ordinary skill in the art. The materials, methods, and examples 25 are illustrative only, and not limiting.
The invention described herein provides a straightforward approach for the correction of a sickle cell anemia defect, and thus has important therapeutic value. Other features and advantages of the invention 30 will be apparent from the following description of the preferred emhoA;ments thereof, and from the claims.

Detailed Description The drawings are first described.

W095/00657 2 1 b 5 6 7 9 PCT~S94/06901 Drawin~s Fig. lA is an electron micrograph of an Hb S fiber and a schematic representation of an Hb S fiber. Fig. lB
is an illustration of the structure of the Hb S fiber.
5 Each circle represents a Hb S tetramer. The fiber is compoco~ of seven pairs of double stranded polymers; two double stranded polymers (4 strands) form the inner core and five double stranded polymers (10 strands) form the outer sheath. Two types of contacts occur between Hb S
10 tetramers incorporated into fibers. Contacts along the long axis of the fiber are termed axial contacts, while contacts along the sides of tetramers are lateral contacts. The ~6 valine plays a critical role in the lateral contact by interacting with the hydrophobic 15 residues ~85 phenylalanine and ~88 leucine. An important axial contact is the interaction of the ~22 glutamic acid with the imidazole group of the ~20 histidine on an adjacent tetramer.
Fig. 2 is an illustration of a lateral contact in 20 the double stranded Hb S polymer. This contact forms when the ~6 valine of sickle hemoglobin interacts with a hydrophobic pocket on an adjacent tetramer. This hydrophobic pocket consists primarily of the residues ~85 phenyl A 1 Ani no (phe) and ~88 leucine (leu)- These two 25 residues are essential for correct positioning of the heme moiety and cannot be mutated. However, a threo~i no (thr) residue at position 87 can be replaced by a glutamine (gln), shown in red. The longer side chain of the glutamine prevents the ~6 valine from interacting 30 with the hydrophobic pocket.
F~g. 3 is an illustration of an axial contact in the double stranded Hb S polymer. The side ch~; nc of the amino acids ~17 lysine (lys), ~19 asparagine (asn), and ~22 glutamic acid (glu) project to form a surface which 35 stabilizes the axial contact. In several of the anti-WO95/~K57 2 1 6 5 6 7 9 PCT~S94/06901 ~ickling hemoglobins, the ~22 glutamic acid is replacedby an alanine residue (ala). This residue fails to interact with the positively-charged histidine from the neighboring tetramer and thus disrupts the axial contact.
5 The new alanine residue is shown in light blue.
Fig. 4 is an illustration of 5' HS 1-5 ~1 and HS
1-5 ~AS constructs. One hundred kilohAces of the human ~-globin locus and 35 kilohA~ee of the human ~-globin locus are illustrated. Cosmids contAin;ng HS 1-5 ~1 and HS 1-5 10 ~AS were constructed by fusing either the ~1 gene or a recombinant anti-sickling ~-globin gene downstream of the ~-globin locus control region (LCR). The 26 kb inserts were purified from vector sequences, mixed at a 1:1 molar ratio (final DNA conc~ntration was 2 ng/~l), and co-15 injected into fertilized mouse eggs. Transgenic linesdisplaying high-level, hAlAnceA expression of the transgenes were establi~heA.
FigQ. 5A-5D are graphs of chromatographs of hemolysates and HPLC-purified anti-sickling hemoglobins.
20 Fig. 5A is a graph of a chromatograph of hemolysate obtained from transgenic mice expressing Hb AS1.
Hemoglobins were separated by non-d enaturing HPLC.
Twenty eight percent of the hemoglobin in erythrocytes of these animals is recombinant human ~Asl. Fig. 5B is a 25 graph of denaturing HPLC analysis of ~Asl purified from the hemolysate shown in Fig. 5A. Purification was performed by preparative isoelectric focusing (IEF).
Approximately 10% of the ~-globin çhAinc were of murine origin. Fig. 5C is a graph of a chromatograph of 30 hemolysate obtained from transgenic mice expressing Hb AS2. Hemoglobins were separated by non-denaturing HPLC.
Eighteen percent of the hemoglobin in the erythrocytes of these animals is recombinant human ~As2. Fig. 5D is a graph of denaturing HPLC analysis of ~As2 purified from 35 the hemolysate shown in Fig. 5C. Purification was W095/~657 2 1 6 5 6 7 q PCT~S94/06901 performed by preparative IEF. This hemoglobin lacks any contaminating murine globins. Hemoglobins purified by preparative IEF were used in all subsequent experiments.
Fig8. 6A-6D are oxygen equilibrium curves (OECs) 5 for purified human anti-sickling hemoglobins. Fig. 6A is an OEC curve for Hb ASl at pH 7.0 in 0.1 M potassium phosphate (KPO4) buffer at 20C. Fig. 6B is an OEC curve for Hb ASl under the same conditions as those described in Fig. 6A, with the addition of 2 mM 2,3-10 diphosphoglycerate (DPG). Fig. 6C is an OEC curve for HbAS2 at pH 7.0 in 0.1 M KP04 buffer at 20C. Fig. 6D is an OEC curve for Hb AS2 under the same conditions as those described in Fig. 6C with the addition of 2 mM 2,3-DPG.
Figs. 7A-7B are graphs showing polymerization delay times for deoxygenated mixtures of human hemoglobins. Fig. 7A shows delay times for hemoglobin mixtures contAin~g 100% Hb S or 75% Hb S, together with 25% Hb A, Hb ASl, Hb AS2, or Hb F. Curves were 20 determined at a hemoglobin concentration of 60 mg/dl using the temperature jump method (Adachi et al., J.
Biol. Chem. 254:7765, 1979). The delay time is an indication of the ability of a hemoglobin to disrupt the polymerization of Hb S. The delay time of Hb ASl is 25 between that of Hb A and Hb F, while the delay time of Hb AS2 is similar to that of Hb F at this hemoglobin concentration. Fig. 7B shows delay time vs. hemoglobin concentration. The progression of the plots from left to right demonstrates the increased Hb concentrations which 30 are required for polymerization to occur in the pr~C~nc~
of the various non-S hemoglobins. The delay time plots for Hb AS2 and Hb F overlap, indicating that the anti-polymerization activities of Hb AS2 and Hb F are virtually identical.

W095/~657 PCT~S94/06901 Fig. 8 is an illustration of a retroviral vector useful for the production of anti-sickling hemoglobin.

Anti-Sickling ~-alobin Genes Desiqned to Inhibit Hb S
Polymerization Recombinant hemoglobins of the invention which contain anti-sickling mutations can be used to inhibit Hb S polymerization, and thus facilitate therapies for sickle cell anemia. In particular, the glutamic acid to valine change at the 6th position of the ~8 polypeptide 10 creates a non-polar surface that readily interacts with a natural hydrophobic pocket in the ~ chain of a C~conA
tetramer. This natural pocket is formed primarily by a phenylalanine (phe) at position 85 and a leucine (leu) at position 88. This interaction leads to the formation of 15 the complex 14-stranded fibers described above, and illustrated in Figs. lA-lB (Bunn et al., Hemoglobin:
Molecular. Genetic and Clinical As~ects, 1986, W.B.
Saunders, Philadelphia).
The structure of the fiber that forms in sickle 20 erythrocytes was derived from X-ray diffraction studies of Hb S crystals (Edelstein, J. Mol. Biol. 150:557, 1981). Hb S tetramers are composed of two ~-globin subunits (~2) and two ~-globin subunits (~2)~ and form characteristic double stranded fibers. Interactions 25 along the long axis of the fiber are termed axial contacts, while interactions along the sides of tetramers are lateral contacts (Fig. lB; Bunn et al., Hemoalobin:
Molecular Genetic. and Clinical As~ects. (W.B. Saunders, Philadelphia, 1986)). The ~6 valine plays a critical 30 role in the lateral contact by interacting with the hydrophobic residues ~85 phenylalanine and ~88 leucine (Fig. 2). Accordingly, to interfere with detrimental Hb S polymerization, this interaction and, thus, hydrophobic pocket formation should be disrupted. Because Hb A (~2~2) 35 has these same hydrophobic residues and is readily WO95/~K57 PCT~S94/06901 incorporated into sickle fibers, it cannot be used for this ~u~ose. Moreover, although disru~tion of this pocket represents the best approach for inhibiting Hb S
polymerization, certain strategies have detrimental side 5 effects. For example, although amino acid substitutions at ~85 phe and ~88 leu would interfere with pocket formation, these amino acids are also important for correct positioning of the heme moiety, and cannot be mutated without severely altering oxygen affinity (Dickerson et al., Hemoglobin: Structure. Function Evolution. and Pathology. (Benjamin/Cummings, Menlo Park, CA, 1983)).
A better approach for inhibiting Hb S
polymerization is depicted in Fig. 2 which shows a 15 computer model of a ~87 threon;ne (thr) to glutamine (gln) substitution that disrupts the hydrophobic pocket, without inhibiting ~-globin function (Perutz et al., Nature 219:902-909, 1968; Computer graphics generated using an Evans and Sutherland PS300 system rlln~;~g the 20 package FRODO (Jones, Meth. Enz. 115:157, 1985)). The long side chain of glutamine prevents the ~6 Val from interacting with the hydrophobic pocket. Human y- and ~-globin polypeptides both have such a glutamine at position 87, and both Hb F (~2Y2) and Hb A2 (~2~2) have 25 potent anti-sickling activity (Nagel et al., Proc. Natl.
Acad. Sci., USA 76(2):670-672, 1979). Another naturally occurring human hemoglobin, designated Hb D ThA~An, also has anti-sickl;ng activity (Watson-Williams et al., Nature 205:1273, 1965). This hemoglobin has a lysine at 30 position 87 and its long side chain also projects across the hydrophobic pocket and inhibits interactions with the ~6 Val.
Preferably, to produce a recombinant anti-sickling hemoglobin, the mutations described above (which 35 interfere with a major lateral contact) are combined with WO9~/~K57 2 1 6 5 6 7 9 PCT~S94/06901 a second mutation which interferes with an axial contact.
One such axial contact-disrupting mutation is shown in Fig. 3. The side chains of the amino acids lysine-17 (lys), asparagine-19 (asn), and glutamic acid-22 (glu) 5 project to form a surface which stabilizes the axial contact with another sickle hemoglobin tetramer (Dickerson et al., Hemoqlobin: Structure Function.
Evolution. and Patholoov. (Benjamin/Cummings, Menlo Park, CA, 1983)). Although mutations at residues 17 or 19 are 10 detrimental, amino acid 22 can be mutated from glutamic acid to alanine (ala) without an alteration in hemoglobin function (Bowman et al., Biochemical and Biophysical Research Communications 26(4):466-470, 1967; Bunn et al., Hemoglobin: Molecular Genetic and Clinical Aspects.
(W.B. Saunders, Philadelphia, 1986)). The negative charge of the glutamic acid side chain at this position plays a key role in stabilizing the axial contact because it interacts with the positively charged imidazole group of a histidine at position 20 in the ~ chain of the 20 neighboring tetramer. The shorter nonpolar AlAnin~ side chain fails to stabilize this interaction, thus disrupting the axial contacts between sickle hemoglobin tetramers. The substituted Al A~; ne residue is shown in light blue in Fig. 3. Hb AS2 contains a glutamine at 25 position 87 together with an alanine at position 22. Hb AS1 has the same ~22 Al ~n; ne and asparagine at ~80 is replaced by lysine. This ~80 lysine significantly inhibits sickl ing when present as a single site mutation in Hb A (Nagel et al., Nature 283:832, 1980). The 30 following 27-mer oligos were used for mutagenesis at the indicated amino acids in ~-globin: ~22, GTGAACGTGGATGC~llGGTGGTGAG (SEQ ID NO: 1); ~80, GCTCACCTGGACAAGCTCAAGGGCACC (SEQ ID NO: 2); ~87, GGCACCTTTGCCCAGCTGAGTGAGCTG (SEQ ID NO: 3).

wo 9~,~657 2 1 6 5 6 7 9 PCT~S94tO6901 Another anti-sickling mutation in the human ~-globin gene useful in the invention is the Hb G Szuhu mutation, a ~80 asn to lys mutation which has significant anti-sickling activity (Nagel et al., Proc. Natl. Acad.
5 Sci. USA 76(2):670-672, 1979), but which does not impair hemoglobin function (Kaufman et al., Human Heredity 25:60-68, 1975). This mutation is preferably combined with the ~22 glu to ala mutation described above.
Alternatively, an a-globin mutation may be 10 utilized to inhibit Hb S polymerization. One example of such an ~-globin mutation is provided by the hemoglobin designated Hb Montgomery (Brimhall et al., Biochim.
Biophys. Acta. 379(1):28-32, 1975), which contains an ~48 leucine to arginine mutation. The 54 year old patient 15 from which this mutation was isolated was homozygous for ~, but had no history of painful sickle cell crises, jaundice, leg ulcers, or stroke, and was only mildly anemic (Prchal et al., Am. J. Med. 86(2):232-236, 1989).
Anti-si~kling hemoglobin AS3 combines the 20 mutations at ~22 and ~87, which are present in anti-sickling hemoglobin AS2, with an additional mutation which lowers the oxygen affinity of the recombinant hemoglobin. The goal is to produce an anti-si~kl;~g hemoglobin which delivers oxygen to tissues prior to 25 sickle hemoglobin (Hb S). We have termed this concept "preferential deoxygenation." If the anti-sickling hemoglobin delivers oxygen preferentially, Hb S will remain oxygenated and, therefore, will not polymerize.
The mutation which was selected to lower the 30 oxygen affinity of the anti-sickling hemoglobin is a change from asparagine to lysine at position 108 of the ~-globin chain. This is the mutation which is present in the naturally-occurring Hb Presbyterian (Moo-Penn et al., FEBS Letters 92:53-56, 1978). Hb AS3 has the following 35 three mutations: (1) ~22 glutamic acid to Al~n;~e, (2) WO95/00657 2 1 6 5 6 7 9 PCT~S94/06901 ~87 threonine to glutamine, and (3) ~108 asparagine to lysine.
Two additional anti-sickling hemoglobins, AS4 and AS5, have been made which combine the mutations present 5 in Hb AS2 at ~22 and ~87, with additional mutations which cause the ~-globin subunit to become more negatively charged. In red blood cells, surface charge is a key determinant of the ability of ~-globin and ~-globin monomers to associate with each other to form dimers (Bunn, Blood 69:1-6, 1987). The alpha subunit is somewhat positively-charged, while the beta subunit is somewhat negatively-charged. By increasing the negative charge on the ~-globin subunit, it is possible to increase its affinity for the ~-globin subunit.
15 Introduction of an additional negative charge in the anti-sickling hemoglobin will provide ~AS polypeptides with a competitive advantage for interacting with ~-globin polypeptides. Consequently, ~2~AS2 tetramers will form more efficiently than ~2~S2 tetramers.
Anti-sickling hemoglobins Hbs AS4 and AS5 combine the mutations present in AS2 with a mutation which increases the negative charge on the ~-globin subunit.
One mutation which increases the negative charge on the ~-globin subunit but which does not affect the normal 25 functioning of the hemoglobin molecule is a change from lysine to glutamic acid at position 95. This mutation occurs naturally and is known as Hb N-Baltimore. The resulting change in charge is -2, since a positively-charged lysine is replaced by a negatively-charged 30 glutamic acid. This chznge in charge also allows Hb AS4 and Hb S to be distinguished by isoelectric focusing. Hb AS4 has the following three mutations: (1) ~22 glutamic acid to ~ ne~ (2) ~87 threon;ne to glutamine, and (3) ~95 lysine to glutamic acid.

W095/00657 2 1 6 5 6 7 9 PCT~S94/06901 An additional mutation which occurs naturally and which is known to increase the ability of the ~-globin subunit to compete for the ~-globin subunit is known as Hb J-Baltimore. This mutation consists of a change from 5 glycine to aspartic acid at position 16 of the ~-globin subunit. While this mutation adds only one additional negative charge to the ~-globin chain (compared to the two negative charges added by the N-Baltimore mutation described above), the location of the negative charge is 10 significant. In fact, Hb J-Baltimore competes even more effectively than Hb N-Baltimore for the ~-globin subunit.
Hb AS5 has the following three mutations~ 16 glycine to aspartic acid, (2) ~22 glutamic acid to alanine, and (3) ~87 threonine to glutamine.
The invention includes anti-sickling hemoglobins that contain any combinations of the individual mutations described above. For example, the ~108, ~95, and ~16 mutations may occur either alone, in combination with the ~22 mutation, or in combination with the ~22 mutation and 20 either the ~80 or either of the above-described ~87 mutations.
Mutaqenesis of Human ~- and B-globin Genes Mutations may be i..L~Gd~ced into the normal human ~- and ~-globin genes by site-directed mutagenesis. For 25 example, a 3.8 kb BglII-EcoRI fragment containing the human ~-globin gene or a 4.1 kb HpaI-XbaI fragment containing the human ~-globin gene may be cloned into the pSELECT plasmid (Lewis et al., Nucl. Acids. Res. 18:3439-3443, 1990; pSELECT is available from the American Type 30 Culture Collection, Rockville, Maryland, ATCC~ 68196) using stAn~Ard methods (see e.g., MAn;Atis et al., 1989, Molecular Clonin~: A LaboratorY Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
Oligonucleotide mutagenesis is performed, e.g., as 35 described by Lewis et al. (Nucl. Acids. Res. 18:3439-W095/00657 2 1 6 5 6 7 9 PCT~S94/06901 3443, l99O). In this procedure, an oligonucleotide which corrects a mutation in the ampicillin resistance gene in the pSELECT plasmid is used simultaneously with one or more oligonucleotides designed to create mutations in the 5 globin gene insert.
Briefly, E. coli (JMlO9; ATCC# 53323) contAining the pSELECT plasmid with globin gene inserts are infected with helper phage (M13K07). After growing the culture overnight (about 12-16 hours), phage obt~ine~ from the 10 supernatant are extracted with phenol:chloroform, and single-stranded DNA is isolated by st~n~Ard methods.
Oligonucleotides cont~;ning each of the mutations are annealed to single-stranded DNA together with the wild-type ampicillin oligonucleotide, and these primers are 15 extended with Klenow for about 9O minutes at 37C.
Double-stranded DNA is transformed into E. coli (BMH 71-18 mutS), and the culture is grown overnight in L- broth containing 75 ~g/ml ampicillin. DNA obtained from rapid lysis preparations of these cultures is transfected into 20 E. coli (JM109), and colonies are selected on ampicillin plates (75 ~g/ml). Double-stranded DNA obtained from rapid lysis preparations of these colonies is se~nc~
(Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) using oligonucleotide primers located upstream of 25 the mutagenic oligonucleotides. Mutants are clearly identified by comparison to wild-type sequence.
Construction of Cosmid Clones The DNA constructs used to produce transgenic animals that synthesize high levels of anti-sickling 30 hemoglobins are illustrated in Fig. 4. Constructs used for microinjection are as described by Behringer et al.
(Science 245:971, 1989), except that the gene for sickle hemoglobin is replaced with genes encoding anti-sickling hemoglobins. Mutations are introduced into the human ~-35 globin gene by site-specific mutagenesis, as described WO95/00657 2 l 6 5 6 7 9 PCT~S94/06901 above, and the mutant sequences are inserted downstream of a 22 kb DNA fragment containing the DNAse hypersensitive sites 1-5 (5' HS 1-5) of the ~-globin LCR
(Lewis et al., Nucleic Acids Res. 18:3439, 1990), as 5 described in further detail below.
In order to construct cosmid clones containing mutant ~- and ~-globin genes, the mutant genes are excised from pSELECT plasmids and subcloned into "right arm" plasmids containing a Cos site. Specifically, a 1.2 lO kb NcoI-XbaI fragment from the ~-globin pSELECT plasmids and a 1.4 kb ClaI-BamHI fragment from the ~-globin pSELECT plasmids are inserted into right arm plasmids in place of the corresponding a- and ~-globin gene wild-type fragments. The ~-globin right arm plasmids are digested 15 with ClaI and MluI, and 4.8 kb fragments containing mutant ~-globin genes which are linked to Cos sites are purified by agarose gel electrophoresis. The ~-globin right arm plasmids are digested with ClaI and HindIII, and 6.5 kb fragments contA;ning mutant ~-globin genes 20 which are linked to Cos sites are purified similarly.
Cosmids containing these fragments are constructed in four way ligations (Ryan et al., Genes Dev. 3:314-323, 1989). The left arms are 9.0 kb MluI-SalI fragments obt~ine~ from the cosmid vector pCV001 (Lau ~ al., Proc.
25 Natl. Acad. Sci. U.S.A. 80:5225-5229, 1983). This fragment contains a Cos site, an ampicillin resistance gene, a ColE1 origin and the SVneo gene. The two internal fragments are a 10.7 kb SalI-KpnI fragment contAining DNase I super-hypersensitive (HS) sites V, IV
30 and III, and a 10.9 kb RpnI-ClaI fragment contAining HS
II and I. The four fragments are ligated together in a 2:1:1:2 molar ratio of vector arms to inserts and packaged (Packagene; Promega, Madison, WI). E. coli ED8767 is infected with the packaged cosmids and is 35 plated onto ampicillin plates. Large scale cultures of WO95/00657 2 1 6 5 6 7 9 PCT~S94/06901 ampicillin resistant colonies are grown, and cosmids are prepared by st~n~rd procedures.

Trans~enic animal assays - Characterization of anti-sicklinq hemoqlobins The effects of anti-sickling hemoglobin can be analyzed using transgenic animals. Cosmid DNA is prepared by stAnA~rd procedures. HS I-V ~ and HS I-V
cosmids cont~;ning the mutations described above are either injected directly into fertilized mouse eggs, or 10 the constructs are digested with SalI and insert DNA is separated from plasmid DNA by agarose gel electrophoresis prior to injection. The injected eggs and transferred to pseudopregnant foster mothers (Brinster et al., Proc.
Natl. Acad. Sci. USA 82:4438-4442, 1985), and transgenic 15 progeny are identified by Southern blot hybridization of tail DNA. Similarly, large animal eggs can be injected with the same constructs and transferred to foster mothers as described by Pursel et al. (Science 244:1281-1288, 1989). Typically, human ~- and ~-globin genes are 20 cloned into expression vectors designed to direct high levels of ~- and ~-globin synthesis in erythroid cells of transgenic animals. These constructs are co-injected into fertilized mouse eggs and expression is analyzed in transgenic animals that develop.
Blood collected from transgenic animals is washed with saline, and hemolysates prepared as described by Ryan et al. (Science 245:971-973, l99O). Hemoglobin is analyzed on isoelectric focusing (IEF) gels (Ryan et al., Science 245:971-973, 1990) to demonstrate that a complete 30 human hemoglobin is formed in adult erythrocytes, and to identify transgenic animals which synthesize high levels of human hemoglobin (Ryan et al., Science 247:566, 1990;
Behringer et al., Science 245:971, 1989). Human hemoglobin bands are excised from IEF gels and analyzed 35 on urea cellulose acetate strips to demonstrate that the WO9~/00657 2 1 6 5 6 7 9 PCT~S94/06901 human hemoglobin band is composed of human ~- and ~-globin polypeptides. It is noted that if human hemoglobin is difficult to separate from endogenous hemoglobins, mutations that increase or decrease the 5 isoelectric point (pI) of human hemoglobin can be introduced into the ~- and ~-globin genes. Increases in pI are accomplished by introducing basic (positively charged) amino acids into the protein, while decreases in pI are accomplished by introducing acidic (negatively lO charged) amino acids. These charged amino acids are introduced at positions which do not disturb the structure or function of the protein. Oxygen equilibrium curves (OECs) of human hemoglobin purified from the transgenic mice are determined as described by Ryan et 15 al. (Science 247:566-568, 1990).
The anti-sickling properties of the AS hemoglobins (purified from erythrocytes of the above-described transgenic animals) can be quantitated by in vitro solubility assays as described, e.g., by Benesch et al.
(J. Biol. Chem. 254:8169, 1979). Briefly, the anti-sickling hemoglobin is mixed with Hb S. The solution is cooled to OC, deoxygenated, and then incubated at 30C
for 2 to 3 hours. Insoluble polymers are pelleted by ultracentrifugation, and the concentration of hemoglobin 25 in the supernatant is determined spectrophotometrically.
The solubility of mutant hemoglobin/Hb S mixtures is compared with Hb A/Hb S and Hb F/Hb S solutions.
Retroviral Vectors Desi~ned To Correct the Sickle Defect The anti-sickling hemoglobin genes described 30 herein may be used to correct a sickling defect by gene therapy. Such techn;ques are first ~ested in an animal model, for example, mice, but similar techn;ques may be used to treat other mammals, including humans. A
description of a retroviral vector useful for WO95/~657 2 1 6 5 6 7 9 PCT~S94/06901 transferring anti-sickling hemoglobin genes to a mammal now follows.
As a first step toward gene therapy, the anti-sickling ~-globin genes described above are preferably 5 inserted into a retroviral vector such as that illustrated in Fig. 4. This vector is of a small size to optimize the viral titers obtained. To construct such a small vector, a minimum ~S 2 region (Caterina et. al., Proc. Natl. Acad. Sci. USA 88:1626-1630, 1991) of the 10 globin LCR is inserted upstream of the ~-globin gene;
this 1.1 kb KpnI-XbaI fragment cont~;ning HS 2 retains sequences required for both enhancer and domain opening activity, facilitating high level expression of downstream ~-globin genes with minimum size. In 15 addition, the vectors include mini-globin genes which contain only 200 bp of 5' flanking sequence and 150 bp of 3' flanking sequence, and only 100 bp of IVS 2. The mini-globin genes, although small, contain all of the se~lenceC nececs~ry for high level expression, including 20 the TATA (-35), CCAAT (-70) and CACCC (-100) boxes (Antoniou et al., Genes Dev. 4:1007-1013, 1990; Antoniou et al., Genes Dev. 4:1007-1013, 1990). They also contain all sequences required for correct splicing of the ~-globin second intron, including the splice donor, splice 25 acceptor, and branch point sequences.
Another feature of these constructs is the use of a deleted SV-Neo region (SN) of LXSN (Miller et al., Biotec-hn;ques 7(9):980-990, 1989); this deletion removes 1.5 kb of DNA and significantly re~nc~c the size of the 30 construct. Although these viruses cannot be easily titered by conventional means, viral titers can be estimated by Southern blot hybridization of NIH 3T3 cells that are infected with supernatants from packaging cells lines. Briefly, to carry out such an assay, the 35 constructs described above are co-transfected with an SV-W095/00657 2 1 6 5 6 7 9 PCT~S94/06901 Neo plasmid into an ecotropic and/or amphotropic packaging cell line (for example, E86 and PA317) (Markowitz et al., Virology 167(2):400-406, 1988;
Markowitz et al., J. Virol. 62(4):1120-1124, 1988; Miller 5 et al., Mol. Cell Biol. 6:2895-2902, 1986), and colonies of G418 resistant cells are isolated. Undiluted and serially diluted supernatants from these colonies are used to infect NIH 3T3 cells; a high titer LXSN virus is used as a control. Southern blot hybridizations with an 10 LX specific probe identify supernatants that efficiently tr~nC~llce intact copies of the retrovirus to 90-100~ of the cultured cells.
If desired, before transfecting retroviral DNAs into packaging cell lines, the constructs may be tested 15 for expression in 16 day fetal liver of transgenic mice as described by Ryan et al. (Genes Dev. 3:3 -323, 1989).
Constructs that are expressed at a high lev are used to produce virus for bone marrow infections.
When a packaging cell line that produces high 20 titer virus (preferably, 106/ml) is obtained, bone marrow from Hb S mice is infected; production of such Hb S mice is carried out as described above using mutant Hb S
hemoglobin transgenes (see, e.g., Ryan et. al., Science 247:566-568, 1990). To facilitate infection, bone marrow 25 from Hb S mice are co-cultured with the packaging line in the pr~C~n~ of IL-3 and IL-6 (Bodine et al., Proc. Natl.
Acad. Sci. USA 86(22):8897-901, 1989). After 48 hours, cells are injected via the tail vein into recipient Hb S
animals that have been lethally irradiated. After a one 30 month recovery period, small aliquots of blood are removed and hemoglobins are analyzed on native IEF gels and denaturing cellulose acetate strips (see, e.g., Behringer et. al., Science 245:971-973, 1989.
Preparative IEF was performed on 4% acrylamide gels with 35 2% Pharmalyte pH 6.7 to 7.7. Bands of hemoglobin were WOg5/~657 PCT~S94/06901 sliced from the gel and eluted in 0.1 M potassium phosphate buffer, pH 7.0). When animals that express human ~-globin at approximately 20% of total ~-globin are obt~ine~, solubility assays are performed to quantitate 5 anti-sickling activity. Also, erythrocytes from these animals are deoxygenated and examined for sickled forms.
Results are compared to control animals that have been transplanted with Hb S marrow infected with LXSN virus only.
Mutant hemoglobins shown to inhibit sickling in Hb S mice are then included in the appropriate mammalian retroviral vector and introduced into a mammal of choice, generally as described above. Retroviral vectors, or other viral vectors with the appropriate tropisms for lS blood cells, may be used as gene transfer delivery systems for the anti-sickling hemoglobin gene. Numerous vectors useful for this purpose are generally known and have been described (see for example, Miller, Human Gene Therapy 1:5-14, 1990; Friedman, Science 244:1275-1281, 20 1989; Anderson, Science 256:808-813, 1992; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biot~chnology 1:55-61, 1990; Cornetta et al., Nucleic Acid Rec~rch and Mol~c~ r Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984;
25 Moen, Blood Cells 17:407-416, 1991; and Miller et al., Biotech~iques 7:980-990, 1989). Retroviral vectors are particularly well developed and have been used in a clinical setting (see, for example, Rosenberg et al., N.
Engl. J. Med. 323:370, 1990). Preferably, the anti-30 sickling hemoglobin genes are introduced by retroviraltransfer into a sample of a patient's bone marrow stem cells (also as described herein and in Miller, 1990, su~ra; Friedman, 1989, supra; Anderson, 1992, su~ra;
Eglitis et al., 1988, supra; Tolstoshev et al., 1990, 35 supra; Cornetta, 1987, su~ra; Anderson, 1984, supra;

W095/~657 2 1 6 5 6 7 9 PCT~S94/06901 Moen, 1991, su~ra; Miller et al., 1989, suPra; and, Rosenberg et al., 1990, supra).

Example: Characterization of anti-sicklinq hemoglobins AS1 and AS2 ~roduced in transgenic mice Transgenic lines expressing ASl or AS2 were established, and hemolysates obtained from several animals were analyzed by anion exchange high performance liquid chromatography (HPLC) to quantitate the amounts of human, mouse, and hybrid hemoglobins (Ip et al., Anal.
10 Biochem. 156:348, 1986; Hemoglobin tetramers were separated by anion exchange HPLC utilizing a Synchropak AN 300 (4.6 mm x 25 mm) column (SynChrom, Lafayette, IN)). Figs. 5A and 5C show that 28% of total hemoglobin was Hb AS1 in one ~Asl transgenic line, and 18% of total 15 hemoglobin was Hb AS2 in one ~AS2 transgenic line.
Hemoglobins AS1 and AS2 were isolated by preparative IEF
(Behringer et al., Science 245:971, 1989) and the purity of the human hemoglobins was assessed by denaturing reverse phase (HPLC) which separates the ~- and ~-globin 20 s~hlln;ts (Adachi et al., J. Chromat. 419:303, 1987.
Mouse and human globins were separated by RP-HPLC using a Dionex Series 4500i HPLC system (Sunnyvale, CA).
Approximately 25-30 ~g of hemoglobin was injected into a Vydac C4 reversed phase column (4.6 mm x 250 mm;
25 Hibernia, CA) and eluted with a linear gradient of acetonitrile and 0.3% trifluoroacetic acid as described in Shelton et al., J. Liq. Chrom. 7:1969, 1g77). Figs.
5B and 5D show that Hb ASl was approximately 90% pure, while Hb AS2 was purified to homogeneity.
The oxygen equilibrium curves (OEC) for purified Hb AS1 and Hb AS2 are illustrated in Figs. 6A and 6C
(~cAkllra et al. in OxYqen Trans~ort in Red Blood Cells, C. Nicolau Ed. (Pergamin, New York, 1986). Oxygen e~uilibrium curves were measured with a Hemox Analyzer (TCS, Southampton, PA). The OEC were determined in 0.1 M

W095/00657 2 1 6 5 6 7 9 PCT~S94/06901 potassium phosphate buffer, pH 7.0 at 20C). These sigmoidally shaped curves demonstrate the normal cooperativity of oxygen binding (Figs. 6A and 6C). The P50 value, which measures the partial pressure of oxygen 5 at which hemoglobin is half-saturated, was determined for Hb AS1 and Hb AS2 and compared with Hb A and Hb F (Table 1). The P50 for Hb AS1 is slightly elevated, but within the normal range, and this hemoglobin responds normally to the allosteric effector 2,3-diphosphoglycerate (2,3-10 DPG); that is, oxygen affinity is decreased in thepresence of 2 mM 2,3-DPG (Fig. 6B). The P50 for Hb AS2 is slightly lower than normal (6.7 mm Hg) but 2,3-DPG raises this value to 8.4 mm Hg. The oxygen affinity of Hb AS2 is functionally equivalent to Hb F in the presence of 2 15 mM 2,3-DPG (Fig. 6D) and, therefore, Hb AS2 should adequately bind and deliver oxygen in vivo.

Table 1. P50 values for recombinant and naturally-occurring human hemoglobins Sample P50 (mm Hg) without DPG with DPG
Hb AS1 10.5 15.0 Hb AS2 6.7 8.4 Hb A 8.7 13.3 Hb F 8.8 10.0 Anti-~ic-~l ;ng Propertie~ of A81 and A~2 hemoglobins Hb S (100%) or mixtures of Hb S (75%) and Hb A, ASl, AS2, or F (25%) were deoxygenated and polymerization 30 as a function of time was measured spectrophotometrically as the temperature of the hemoglobin solution was raised from 0C to 30C (Adachi et al., J. Biol. Chem. 254:7765, 1979; Adachi et al., J. Biol. Chem. 255:7595, 1980;

W095/00657 PCT~S94/06901 Kinetics of polymerization were determined in 1.8 M
potassium phosphate buffer. Polymerization was initiated using the temperature jump method in which the t~mpe~ature of deoxygenated hemoglobin solutions is - 5 rapidly changed from 0C to 30C and the time course of aggregation is monitored turbidimetrically at 700 nm).
Fig. 7A shows that Hb S polymerizes relatively rapidly and that Hb A, AS1, AS2, and F delay Hb S polymerization to different extents. Hb ASl inhibits Hb A
10 polymerization more efficiently than Hb A; however, Hb AS1 inhibits much less effectively than Hb F which is known to inhibit sickling in vivo at a 3:1 ratio (Noguchi et al., New Eng. J. Med. 318:96, 1988). Finally, Hb AS2 inhibits Hb S polymerization at approximately the same 15 level as Hb F. This result strongly suggests that Hb AS2 will inhibit Hb S polymerization in vivo if expression of AS2 at a level of 25~ of total hemoglobin can be achieved.
The delay times determined in Fig. 7A were all 20 measured at a cQnc~ntration of 60 mg/dl. Fig. 7B shows the results of similar experiments performed at variable concentrations of total hemoglobin. The ratio of Hb S to Hb A, ASl, AS2 or F in all of these experiments was 3:1.
In this figure the log of the reciprocal of the delay 25 time and the log of hemoglobin conc~ntration are plotted.
As reported previously (Hofrichter et al., Proc. Natl.
Acad. Sci. USA 71:4864, 1974; Wishner et al., J. Mol.
Biol. 98:179, 1975; Crepeau et al., Nature 274:616, 1978;
Dykes et al., J. Mol. Biol. 130:451, 1979; Padlan et al., 30 J. Biol. Chem. 260:8280, 198; and Eaton et al., Blood 70:1245, 1987), an empirical relationship between delay time and hemoglobin concentration can be described by the following equation: 1/td=ySn, where S=[Hb]total/~Hb]soluble, and y is an experimental 35 constant. The n value is related to the size of nuclei W095/00657 2 1 6 5 6 7 9 PCT~S94106901 formed during polymerization. The n values of the data shown in Fig. 7B are between 2 and 3, which agree well with those shown previously in high phosphate buffer (Adachi et al., J. Biol. Chem. 254:7765, 1979). At 5 higher concentrations of hemoglobin, the delay times for Hb AS2 and Hb F overlap, indicating that Hb AS2 and Hb F
have virtually identical anti-polymerization activity.
The results described above demonstrate that the genetic modification of two surface amino acids in Hb A
lO produces a unique human hemoglobin tHb AS2) that inhibits Hb S polymerization as effectively as Hb F. As discussed above, the ~-globin LCR enhances ~-globin gene expression much more effectively than y-globin gene expression in adult erythroid cells. Therefore ~As2, which is a ~-15 globin gene with the anti-polymerization properties of ~-globin, a useful molecule for future genetic therapy of sickle cell disease.
It is understood that the examples and embodiments described herein are for illustrative purposes only and 20 that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

W095/~657 2 1 6 5 6 7 9 PCT~S94/06901 8EO~RNCB LI8TING
RNF~T- INFORMATION:
ti) APPLICANT: Townes, Tim M
~ ne, Steven L
(ii) TITL_ OF INVENTION: ANTI-SICKLING HEMOGLOBIN
(iii) NUNB_R OF 8EQ~_NC_8: 3 (iV) COP~ r~S_.vENcE 7~nn~RP~
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(A) NAML: Clark, Paul T.
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W095/006S7 2 1 6 5 6 7 9 PCT~S94/06901 ~i) 8EQ~ENCE r~CTERI8TIC8:
~A) LBNGT~: 27 ~B) TYPE: nucleic acid ~C) 8TRAND~nN~R~ single ~D) TOPOLOGY: linear ~ Qu~._~ nK8C~TPTION: SEQ TD NO: 1:

GTGAACGTGG ATGCC~llGG TGGTGAG 27 ~2) INFORNATION FOR ~Qu~NCB IDENTIFICATION N~NBER: 2:
~i) 8EQu~N~ CU~CTBRI8TIC8:
~A) LBNGT~: 27 ~B) TYP~: nucleic acid ~C) 8TRAND~nN~Q: single ~D) TOPOLOGY: linear ~i) 8EQ~BNCB n~P~PTPTION: SEQ ID NO: 2:

~2) INFORNATION FOR 8EQUENCE ID~.~l~lCATION NUNBER: 3:
i) 8EQU~ K ~P~ CTBRI8TIC8:
~A) LBNGTH: 27 ~B) TYPE: nucleic acid ~C) 8TRAND~nN~: single ~D) TOPOLOGY: linear ~si) ~QU~NC~ D~P~TPTION: SEQ ID NO: 3:

What is claimed is:

Claims (15)

1. A recombinant human hemoglobin molecule with anti-sickling activity.

2. The molecule of claim 1, wherein said hemoglobin is .beta.-globin.

3. The molecule of claim 1, having a mutation which disrupts the hydrophobic pocket formed by .beta.-globin amino acids phenylalanine 85 and leucine 88, said mutation leaving intact the correct positioning of the heme moiety.

4. The molecule of claim 1, wherein said molecule comprises:
(a) a glutamine residue at .beta.-globin amino acid 87;
(b) a lysine residue at .beta.-globin amino acid 87; or (c) a lysine residue at .beta.-globin amino acid 80.

5. The molecule of claim 1 or 4, said molecule having an alanine residue at .beta.-globin amino acid 22.

6. The molecule of claim 1, 4, or 5, said molecule having:
(a) a lysine residue at .beta.-globin amino acid 108;
(b) a glutamic acid residue at .beta.-globin amino acid 95; or (c) an aspartic acid residue at .beta.-globin amino acid 16.

7. The molecule of claim 1, said molecule having an arginine residue at .alpha.-globin amino acid 48.

8. Purified DNA comprising a sequence encoding a recombinant anti-sickling human hemoglobin chain.

9. The DNA of claim 8, wherein said hemoglobin is .beta.-globin.

10. The DNA of claim 8, said DNA comprising a sequence encoding a mutation which disrupts the hydrophobic pocket formed by .beta.-globin amino acids phenylalanine 85 and leucine 88, said mutation leaving intact the correct positioning of the heme moiety.

11. The DNA of claim 8, said DNA comprising a sequence encoding:
(a) a glutamine residue at .beta.-globin amino acid 87;
(b) a lysine residue at .beta.-globin amino acid 87; or (c) a lysine residue at .beta.-globin amino acid 80.

12. The DNA of claim 8 or 11, said DNA comprising a sequence encoding an alanine residue at .beta.-globin amino acid 22.

13. The DNA of claim 8, 11, or 12, said DNA
comprising a sequence encoding:
(a) a lysine residue at .beta.-globin amino acid 108;
(b) a glutamic acid residue at .beta.-globin amino acid 95; or (c) an aspartic acid residue at .beta.-globin amino acid 16.

14. The DNA of claim 8, said DNA comprising a sequence encoding an arginine residue at .alpha.-globin amino acid 48.

15. A transgenic non-human mammal all of whose germ cells and somatic cells contain the DNA molecule of claim 8, introduced into said animal, or an ancestor of said animal, at an embryonic stage.